Mitochondria (mt) are the powerhouses of the cell and the main source and scavenger of reactive oxygen and nitrogen species. In recent years, evaluation of mitochondrial function received a broadening attention in studies of many physio-pathological conditions, with increasing relevance in clinical applications. High-resolution respirometry (HRR) provides the state-of-the-art platform for simultaneous measurement of mitochondrial and cell respiration and additional bioenergetic parameters (i.e., hydrogen peroxide production, mitochondrial membrane potential, ADP-ATP exchange, Ca2+ dynamics). Any mitochondrial preparation (mtprep) can be applied, from isolated mitochondria, tissue homogenate, permeabilized muscle fibers, permeabilized cells, to intact cells, to optimally address the variety of research questions. Substrate-uncoupler-inhibitor titration (SUIT) protocols have been evaluated and standardized, since the appropriated design of SUIT protocols is decisive for an accurate evaluation of mitochondrial function and dysfunction. Therefore, we developed the SUIT reference protocol (RP) with the objective to provide a common reference for establishing a comprehensive mt-physiology database. The SUIT-RP consists of two complementary SUIT protocols (RP1 and RP2) which interrogate several mt-pathways converging at the Q-junction, allowing the identification of specific mt-related injuries. Application examples are shown with the SUIT-RP applied in different mtprep including cardiac isolated mitochondria, brain tissue homogenate, cryopreserved cells, permeabilized fibers and human blood cells. In summary, standardized respiratory SUIT protocols combined with additional bioenergetic parameters offer a powerful tool in biomedical research and clinical diagnostics, providing important information about mitochondrial function in health and disease.